“Myocardial protection” has been a hot subject in studies conducted by cardiac medicine and cardiac surgery in recent years. Recent documents showed that, ischemia and hypoxia could cause many changes to the myocardial cells, including overload of calcium in the cells, generation of free radicals, valvular injury, decrease in ATP (adenosine triphosphate, ATP) level, oxygen exhaustion, etc.
The consummation and popularization of cardiac surgical interventions have released numerous patients from pains and improved their life quality. With the increasingly higher requirements of people for myocardial protection, a lot of people have been in fundamental and clinical studies. The myocardial protection in clinical cardiac surgery includes myocardial protection prior, during and after the surgical operation, yet the focus is still on the prevention of ischemia and reperfusion injury in the course of extracorporeal circulation for cardiac arrest. For this purpose, the fundamental and clinical researchers have been aggressively tackling the issues of myocardial protection, which mainly include: (1) the way of perfusion, for example, direct perfusion, inverse perfusion, simultaneous perfusion, intermittent perfusion or continuous perfusion; and whether a blood cell filter is used, and so on; (2) temperature of the perfusate: mainly including normal temperature, low temperature, etc.; (3) components of the perfusate: such as the additional use of the oxygen free-radical scavenging of super-oxide dismutase, reduced glutathione, aprotinin, or puerarin, etc. These measures have, to a certain degree, changed the pathological processes of the myocardial ischemia-reperfusion injury. Some foreign researchers tried to put phosphokinase (Neoton from Italian OUHUI Pharmaceutical Plant) in the perfusate or have patients take trimetazidine dihydrochloride (Vasorel from French Servier), so as to improve the metabolism of cardiac muscle, and elaborated some of their findings from aspects of cell, subcellular structure, oxygen free radicals, energy metabolism, calcium ion (Ca2+) overload and so on. However, these studies either focused only on the supplement of energy or separated the prevention of damage from the defense mechanism of the organism itself, which led to the unsatisfactory clinical outcome in preservation of the cardiac myocardium. Therefore, it is necessary to research and develop more efficacious therapeutic agents for myocardial protection.
In order to interfere with myocardial ischemia and protect heart muscle, many drugs were developed in the past twenty years, such as β-blockers, calcium antagonists, converting enzyme inhibitosr, various oxygen free-radical scavengers etc, but their protective effect on cardiac muscle is not affirmed clinically yet. None of the existing medicine for the treatment of myocardial ischemia can absolutely decrease myocardial infarction and antagonize myocardial ischemia. The alteration in the salivary gland chromosome of the fruit fly Drosophila following a short-term elevation in the temperature was reported in the late 1980s, indicating genetic transcription was activated, which was called heat shock reaction (HSR, Anna Rev Biochem 1986, 55:1151). Thereafter, it was found that the heat shock preconditioning of experienced animals could obviously decrease damage to the myocardium caused by ischemia/perfusion, and it was named “ischemia precondition” (IP) by Marry in 1986. Further research indicated that a new group of proteins, that is heat shock proteins (HSPS), or so-called stress proteins (SP), were induced and synthesized by heat shock of cardiac myocytes. These successive research reports demonstrated that heart muscle itself has strong cellular tolerance; later, it was found that HSPS can be induced and expressed under the heat shock treatment for both cultured cells and the whole organism, such as prokaryotes, eukaryotes, plants, animals and human beings. The features of HSR and HSPS is shown as following: (1) Besides induction by heat shock, HSP could be synthesized by many other factors, such as ischemia, hypoxia, ethanol, heavy metallic salt, myocardial pressure load, drugs and most disease, and could produce “cross tolerance phenomenon”. (2) HSP had high conservatism in structure; for example, 72% of amino acid sequence of HSP70 is identical for fruit fly and saccharomycete, 73% of HSP70 gene is homologous for human and fruit fly, and 78% for HSP90. These structural similarities guaranteed the functional sameness (Burdon: Biochem, J. 1986, 240:313). (3) HSP had an optimum time horizon on myocardial protection, alternatively called “window of opportunity,” and it would not provide protection if a certain time limit was exceeded (Perdriget: Curr. Surg., 1989, 23). (4) HSP existed in the whole biological universe and was present in various cells of organisms of higher animals. Induction of HSPS not only enhances rehabilitation of myocardial function, but also reinforces rehabilitation of myocardial endothelial function and extends the cardioplegic arrest time. The above described function can be used in donor protection of heart transplantation, salvage of myocardium ischemic, preparation of cardioplegic solution for extracorporeal circulation, etc., and it can break through the restriction of traditional drugs and provide a milestone new method that starts from enhancing the induction of self anti-damage potential of in-vivo cells.
Pennica et al (1995) cloned the cardiotrophin-1 (CT-1) gene from myocardial cells, and expressed it in Escherichia coli. It was indicated that CT-1 is a type of cytokine with the function of immunoloregulation, induction of proliferation, and anti-injury of myocardial cells caused by hypoxia and high temperature. It naturally exists in the myocardial cells and can inhibit the apoptosis of myocardial cells. Further research showed that CT-1 could induce HSP expression in cultured myocardial cells and in vivo. The effect of induction is concerned with a cell surface polypeptide named gp130, and the activated gp130 reinforces the expression of HSP70, HSP90 and micromolecule substrate of HSP through NF-IL-6/NF-IL-6β and tyrosine kinase path, thus enhancing the ability of the myocardial cells to tolerate hypoxia and high temperature. Studies also indicated that CT-1 could promote synthesis of structural protein of the myocardial cells, and the increase in the long axis of cells could make contraction stronger. The study of myotrophin of gene recombination is another subject of stimulating factors of the myocardial cells. Parames (1997) demonstrated that myotrophin promotes growth of the myocardial cells and is associated with protein kinase-C. Myotrophin and cardiotrophin may all be cytokines with similar function in the myocardial cells and different paths to activate cellular proliferation, and both show the function of protecting the myocardial cells and promoting proliferation.
Among the available cardiovascular drugs, except for converting enzyme inhibitor which has the function of blocking the generation of growth factors, inhibiting protein synthesis and myocardial hypertrophy, other drugs do not have the function of regulating the growth, differentiation and rehabilitation of cardiac muscles. In recent years, inducing the protection of cardiac muscle cells themselves by pharmacological treatment has been emphasized overseas, such as the research on promoting the myocardial regeneration by transduction gene etc, and the study on cardiotrophin and myotrophin. Moreover, extracellular signals are used to trigger various transmission mechanisms and to regulate and control the proliferation or reconstitution of myocardial and vascular cells. However, all of this research is at the stage of animal testing or preclinical study.
It is clearly demonstrated from aforesaid studies that, in the situation where protection of cardiac muscle for extracorporeal circulation is not consummated yet, it is of great importance to provide a drug that poses no damage to the organism and can protect cardiac muscle prior, during and after surgical interventions. A new approach to explore the prevention and cure of myocardial ischemia and reperfusion injury is also necessary.
ZL94102798 disclosed a growth-stimulating peptide of the myocardial cells and the process for preparation thereof. The process comprises the steps of: the heart of healthy infant mammals other than a human was crushed with mechanical means, deep frozen at −20° C. and heated to 60-100° C. after dissolving in water, then deep frozen at −20° C. and centrifuged at 3000 rpm after being melted, and finally a polypeptide active substance with molecular weight less than 20000 Da was obtained through negative pressure interception column, sterilization, filling, lyophilization and packing.
ZL94102799 disclosed a growth-stimulating peptide of the myocardial cells (GMGSP) that can stimulate DNA synthesis and protein synthesis of primarily cultured myocardial cells, which was isolated from the heart of healthy infant mammals other than a human mammal, and stabilizes at pH 2-9; the biological activity did not change when GMGSP was heated at 95-100° C. for 10 minutes or at 60-70° C. for 30 minutes, but biological activity was lost when being placed in proteolytic enzymes at 37° C. for two hours; a polymer was formed at 22° C.-30° C. in aqueous solution, but biological activity did not have obvious change; biological activity did not change if GMGSP was lyophilized and sealed with 3%-8% mannitol and stored at room temperature for 1.5 years, or at 4° C. for 2 years, or at −20° C. for 3 years; HPLC analysis indicated that the aforesaid GMGSP is composed of four components. The relative peaks and retention times of each component were respectively 10.4% (2.88 minutes), 6.4% (3.93 minutes), 36.3% (5.09 minutes) and 7.3% (7.41 minutes), and each component has biological activity. The molecular weights of two bands displayed by SDS-PAGE analysis were respectively 8500 Da and 10800 Da. The average molecular weight displayed by HPLC analysis was 9800 Da, average molecular weight was 10500 Da, and both components have biological activity.
However, the biologically active peptides described in above-mentioned patents are obtained by a rough separation, purification, the test of activity is also simple, and it fails to provide detailed description for its ingredients, use and efficacy.